Antibiotic roridin L-2 and its use

ABSTRACT

The present invention relates to a novel trichothecene antibiotic, roridin L-2, and a process for the production and the method of using said compound.

SUMMARY AND DETAILED DESCRIPTION

The present invention relates to roridin L-2 which is represented by theproposed structure I and a process for the production and the method ofusing said compound. ##STR1## More particularly, the process relates toa fermentation process for the production of the compound of thisinvention using a roridin L-2 producing strain of an organism closelyresembling Myrothecium roridum Tode. In addition, the invention relatesto pharmaceutical compositions containing the compound of the inventionalone or in combination with other trichothecene derivatives or otherantitumor agents in the treatment of bacterial infections and neoplasticdiseases. A review of the trichothecene antibiotics is found in C. Tamm,"Progress in the Chemistry of Natural Products," 31, 64-117 (1974).

Mild alkaline hydrolysis of roridin L-2 produces verrucarol (II),##STR2## a dihydroxysesquiterpene which is a structural unit of severaltrichothecenes. Roridin L-2 can be readily distinguished from related,previously disclosed trichothecenes such as roridins A, D, E, E-2, H, Jand K by the following characteristic properties exhibited by roridinL-2:

1. an infrared absorption at 1780 cm⁻¹

2. a proton magnetic resonance signal that appears, in deuteratedchloroform solution, as a doublet (J=2 Hz) centered at 4.80 ppmdownfield from tetramethylsilane.

3. three ¹³ C magnetic resonance signals that appear, in deuteratedchloroform solution, at 174.0, 167.4, and 166.5 ppm downfield fromtetramethylsilane.

The above spectral properties are not shown by any of the knowntrichothecenes and clearly establish roridin L-2 as a new member of thetrichothecene family of antitumor antibiotics.

Culture Characterization and Fermentation Processes

In accordance with the present invention, roridin L-2 is produced bycultivating a selected roridin L-2 producing strain of an organism thatclosely resembles Myrothecium roridum Tode under artificial conditionsin a suitable nutrient medium until a substantial quantity of roridinL-2 is formed and isolating this compound in pure form by proceduresdescribed hereinafter.

A new strain of a Myrothecium roridum species, suitable for the purposeof the invention, has been isolated from a sample of soil collected inCleveland, Ohio. It corresponds morphologically to the description ofthis species by N. C. Preston, "Transactions Brit. Mycological Soc.,"26, 158 (1943); and J. C. Gilman, "Manual of Soil Fungi," Second Edition1957, The Iowa State College Press, Ames, Iowa. Cultures of thisorganism have been deposited with the United States Department ofAgriculture, Northern Utilization Research and Development Division,Peoria, Illinois, and are being maintained in their permanent culturecollection as NRRL 12303.

The antitumor antibiotic compound, roridin L-2, is produced by thefungus during aerobic fermentation under controlled conditions. Thefermentation medium consists of suitable sources of carbon, nitrogen,inorganic salts, and growth factors assimilable by the microorganism.Examples of carbon sources are various sugars such as cerelose, lactose,and maltose; starch, dextrin; corn meal; and glycerol. The normalquantity of the carbon sources varies from about 0.5 to 6% by weight,but levels outside of this range can also be used.

The sources of nitrogen can be of organic, inorganic, or mixedorganic-inorganic in nature. The nitrogen sources that can be used inthe culture medium are cottonseed meal, corn germ flour, soybean meal,corn steep liquor, distillers' solubles, peanut meal, fish meal,peptonized milk, and various ammonium salts. The normal amount addedvaries from 0.1 to 3%, but higher amounts are also acceptable.

The inclusion of certain amounts of minerals and growth factors in thefermentation medium is also helpful in the production of roridin L-2.Crude medium ingredients such as distillers' solubles, corn steepliquor, fish meal, yeast products, peptonized milk, and whey containminerals and growth factors. However, inorganic salts such as potassiumphosphate, sodium chloride, ferric sulfate, calcium carbonate, colbaltchloride, and zinc sulfate can be added to the fermentation medium.

The preferred method for producing roridin L-2 by Myrothecium roridin isby submerged fermentation. According to the embodiment of thisinvention, fermentation ingredients are prepared in solution andsterilized by autoclaving or steam heating. The pH of the aqueous mediumis preferably between six to eight. The fermentation medium is cooled toa suitable temperature, between 20°-45° C., and then inoculated with thesuitable culture. Fermentation is carried out with aeration andagitation, and the maximum production of roridin L-2 is usually reachedin about three to eight days.

In the submerged culture method, fermentation is carried out in shakeflask or in stationary vat fermentors. In shake flasks, aeration isbrought about by agitation of the flask which causes mixing of themedium with air. In the stationary fermentors, agitation is provided byimpellers in the form of disc turbine, vaned disc, open turbine, ormarine propeller; and aeration is accomplished by injecting air oroxygen into the fermentation mixture.

The examples which follow illustrate the preferred methods by which theproduct, roridin L-2, of this invention is obtained. The describedprocess is capable of wide variation, and any minor departure orextension is considered as within the scope of this invention.

Fermentation in 200-Gallon Fermentors A. Seed Development

A culture of the organism Myrothecium roridum, preserved in a soil tube,is transferred to CIM 23 agar slants and incubated at 28° C. for sevendays.

    ______________________________________                                        CIM-23 Slant Medium                                                           ______________________________________                                        Amidex corn starch                                                                              10          gm                                              N--Z Amine, type A                                                                              2           gm                                              Beef extract (Difco)                                                                            1           gm                                              Yeast extract (Difco)                                                                           1           gm                                              Cobalt chloride.6H.sub.2 O                                                                      20          mg                                              Agar              20          gm                                              Distilled water   1,000       ml                                              ______________________________________                                    

The microbial growths in two slants are scraped, suspended in distilledwater, and used to inoculate a 30 liter seed fermentor. The seedfermentor is prepared by charging it with 16 liters of ARM 1558A mediumand then autoclaving for 90 minutes at 121° C. and 15 PSI.

    ______________________________________                                        ARM 1558A                                                                     ______________________________________                                        Cerelose                2.0%                                                  Pharmamedia             0.3%                                                  Defatted corn germ flour                                                                              0.1%                                                  Soybean meal            0.1%                                                  CaCO.sub.3              1.0%                                                  K.sub.2 SO.sub.4.7H.sub.2 O                                                                           0.1%                                                  MgSO.sub.4.7H.sub.2 O   0.1%                                                  NaCl                    0.05%                                                 FeSO.sub.4.7H.sub.2 O   0.0001%                                               ______________________________________                                    

Use tap water, no pH adjustment.

B. Production Fermentation

Two 220 gal fermentation tanks are used for producing roridin L-2. Eachof these fermentors contains 160 gal of presterilized ARM 1558 medium.

    ______________________________________                                        ARM 1558 Medium                                                               ______________________________________                                        Cerelose                5.0%                                                  Pharmamedia             0.3%                                                  Defatted corn germ flour                                                                              0.1%                                                  Soybean meal            0.1%                                                  CaCO.sub.3              1.0%                                                  K.sub.2 HPO.sub.4       0.1%                                                  MgSO.sub.4.7H.sub.2 O   0.1%                                                  NaCl                    0.05%                                                 FeSO.sub.4.7H.sub.2 O   0.0001%                                               ______________________________________                                    

Use tap water, no pH adjustment.

Each fermentor is inoculated with about 15 liters of the microbialgrowth from a 30 liter seed fermentor. The inoculated fermentors arestirred at 190 rpm at 30° C. and sparged with 20 CFM of air for 119hours. Dow Corning antifoam "C" is used to control foaming as required.

Production of roridin L-2 in the fermentation broth is assayed vs KB andL1210 tissue cell lines. An aliquot of the fermentation broth is mixedwith growing cultures of KB and L1210 cells to give a final dilution ofthe fermentation broth of 1:100. Activity is expressed as the percentgrowth of the cells relative to the control (no fermentation brothadded). A fermentation broth that gives percent growth values of 0-25%for KB and 0-50% for L1210 is considered active.

The activities of the fermentation broths after 119 hours offermentation are:

    ______________________________________                                        Inhibitory Zone (mm)                                                          Saccharomyces                                                                             Candida  Cytotoxicity (% growth)                                  cerevisiae  albicans L1210       KB                                           ______________________________________                                        16          15       4           0                                            15          17       4           9                                            ______________________________________                                    

Isolation of Roridin L-2

The fermentation beers (a total of 1350 liters) from two 200 galfermentors prepared as described above are combined and mixed vigorouslywith 675 liters of ethyl acetate at pH 6.6 for one hour. Celite 545 (91kg) is then added and the stirred mixture is filtered using a 75.8 cmplate and frame filter press. The filter cake is washed with 245 litersof ethyl acetate followed by 188 liters of deionized water. The filtrateand filter cake washes are combined and allowed to stand to permit theseparation of phases. The upper organic phase amounts to about 660liters; the lower aqueous phase (1520 liters) is separated and mixedwith 340 liters of fresh ethyl acetate. After standing, the layers areseparated and the upper organic layer (356 liters) is added to the firstethyl acetate extract. This combined ethyl acetate extract (1015 liters)is then concentrated in vacuo to 13.2 liters. The concentrated extractis dried using anhydrous sodium sulfate and then filtered. The solventis removed in vacuo to leave approximately 900 g of an oily residue.This product, hereinafter referred to as residue A, is processed by thefractionation method described in the following example to afford a puresample of roridin L-2.

Purification Method

A solution of 25 g of residue A in 25 ml of methylene chloride isfractionated using a Prep LC/System 500 apparatus fitted with one PrepPak 500®/silica gel cartridge (5.7 cm×30 cm) both of which are availablefrom Waters Instruments, Inc., Milford, Mass. After injection of thecharge, the silica gel column is eluted consecutively with 2000 ml ofmethylene chloride, 2500 ml of 75:25 methylene chloride:ethyl acetate,3000 ml of 50:50 methylene chloride:ethyl acetate, 1000 ml of 25:75methylene chloride:ethyl acetate and finally with 1000 ml of ethylacetate. Analysis of each fraction by means of high performance liquidchromatography (HPLC) using a 0.39 cm (I.D.)×30 cm μ Bondapak® (C₁₈silica gel) column, a solvent system consisting of 60:40 methanol:water,and UV detection at 254 nm showed that most of the fractions eluted with50:50 methylene chloride:ethyl acetate contained the desired material,roridin L-2. The retention time of this compound in the above analyticalHPLC system is approximately 3.2 minutes using a flow rate of 2ml/minute. The fractions (2000 ml) containing roridin L-2 are combinedand concentrated in vacuo to leave an oily residue B (4.2 g).

Residue B is combined with 1.8 g of equivalent material obtained from aprevious Prep LC fractionation and dissolved in 21 ml of 70:30methanol:water. One-third portions of this solution are then subjectedto further fractionation by low pressure chromatography over 250 g ofC₁₈ silica gel (Waters Instrument, Inc.), packed in a 4.8 cm (O.D.)×45cm Michel-Miller column (available from Ace Glass Co., Vineland, N.J.)using an eluant consisting of 50:50 methanol:water. Betweenchromatographic runs, the C₁₈ silica gel column is washed with methanoland reequilibrated with 50:50 methanol:water before the next charge isintroduced. Fractions are monitored using the analytical HPLC conditionsdescribed above. The fractions that contain only roridin L-2 arecombined and concentrated in vacuo to remove methanol. The remainingaqueous phase is extracted with two 500 ml portions of ethyl acetate.The organic extracts are combined, washed with 200 ml of water, driedover anhydrous sodium sulfate, and then filtered. Removal of the ethylacetate by concentration of the filtrate in vacuo afforded 3.4 g of pureroridin L-2 as a white solid. The properties which define roridin L-2 asa novel compound are listed below.

PROPERTIES OF RORIDIN L-2

Ultraviolet Absorption Spectrum in Methanol [FIG. 1] ##EQU1##

Infrared Absorption Spectrum in KBr [FIG. 2] Principal absorptions at:3450, 2970, 1782, 1750, 1640, 1600, 1435, 1284, 1180, 1084, 1030, and965 reciprocal centimeters.

Optical Rotation

[α]_(D) =+83.6° (1.0% in chloroform)

Elemental Analysis

1. dried 18 hours at 25° C./0.01 mm

    ______________________________________                                                         % C    % H    % O                                            ______________________________________                                        Calcd. for C.sub.29 H.sub.38 O.sub.9.0.5 H.sub.2 O:                                              64.55    7.29   28.17                                      Found:             64.68    7.12   28.70                                                         64.41    7.15   28.95                                      ______________________________________                                    

2. dried for 2 hours at 100° C./0.1 mm and then for 4 hours at 50°C./0.1 mm

    ______________________________________                                                          % C  % H                                                    ______________________________________                                        Calcd. for C.sub.29 H.sub.38 O.sub.9 :                                                            65.64  7.22                                               Found:              65.21  6.93                                                                   65.36  7.28                                               ______________________________________                                    

Proton Magnetic Resonance Spectrum in CDCl₃ [FIG. 3] Principal signalsat:

(s=singlet, d=doublet, t=triplet, m=multiple) 0.83s, 1.16d (J=6), 1.72s,2.02m, 2.81d (J=4), 3.13d (J=4), 3.5-4.0 (complex signals), 4.80d (J=2),5.49 broad d (J=6), 5.22-6.02 (complex signals), 6.08 dd (J=4,6), 6.60 t(J=11), and 7.60 dd (J=11, 15) parts per million downfield fromtetramethylsilane.

Carbon-13 Nuclear Magnetic Resonance Spectrum in CDCl₃ [FIG. 4]

The principal signals, in parts per million downfield from TMS, are at:

    ______________________________________                                        Signal  Ppm (downfield                                                                              Signal     Ppm (downfield                               Number  from TMS)     Number     from TMS)                                    ______________________________________                                        1       173.95              18       73.46                                    2       167.43              19       69.74                                    3       166.46              20       66.83                                    4       143.37              21       66.34                                    5       140.57              22       65.59                                    6       138.89              23       62.51                                    7       130.53              24       48.86                                    8       118.88              25       48.00                                    9       118.72              26       44.28                                    10      116.67              27       36.19                                    12      85.38               28       29.23                                    13      78.91               29       28.04                                    14      78.42               30       23.19                                    15      77.02         CDCl.sub.3                                                                          31       21.14                                    16      75.62               32       18.44                                    17      75.40               33       6.57                                     ______________________________________                                    

High Performance Liquid Chromatography

Column: μ Bondapak C₁₈ (3.9 mm I.D.×30 cm)

System: 60:40 methanol:water

Flowrate: 2 ml/min

Detection: ultraviolet absorption at 254 nm

Retention time: 3.2 min

Antitumor Activity of Roridin L-2 Against P388 Lymphatic Leukemia inMice

    ______________________________________                                                          T/C* percent                                                Dose              MST                                                         (mg/kg/day)       test 1 test 2                                               ______________________________________                                        16                --     185                                                  8                 157    176                                                  4                 134    132                                                  2                 140    144                                                  1                 132    150                                                  ______________________________________                                         *T/C percent MST = 100 × median survival time in days of                treated/control mice. Values ≧ 130 are considered active. The test     method used is based on that described in Cancer Chemother. Reports 3:        1-87 (part 3), 1972.                                                     

The ID₅₀ of Roridin L-2 against L1210 cells is 0.178 μg/ml. The zonediameter of inhibition of Roridin L-2 against KB cells in agar is 28 mmwhen a 6.4 mm paper disk moistened with a solution containing 100 μg ofRoridin L-2 per milliliter is used; and 12 mm when a 6.4 mm paper diskmoistened with a solution containing 10 μg of Roridin L-2 per milliliteris used.

The antibiotic roridin L-2 can be used for its antimicrobial in the formof pharmaceutical compositions containing roridin L-2 and a compatiblepharmaceutically acceptable carrier. The compositions may also containother active antibacterial and/or antitumor agents. The compositions maybe made up in any pharmaceutical form appropriate for the route ofadministration in question. Examples of such compositions include solidcompositions for oral administration such as tablets, capsules, pills,powders and granules, liquid compositions for topical or oraladministration such as solutions, suspensions, syrups and elixirs, andpreparations for parenteral administration such as sterile solutions,suspensions, or emulsions.

For use as an antibacterial agent, the compositions are administered sothat the concentration of active ingredient is greater than the minimuminhibitory concentration for the particular organism being treated.

We claim:
 1. Antibiotic roridin L-2 having the structural formula##STR3## and characterized by: (a) an infrared spectrum in potassiumbromide having principal absorptions at 3450, 2970, 1782, 1750, 1640,1600, 1435, 1284, 1180, 1084, 1030 and 965 reciprocal centimeters(b)[α]_(D) 23=+83.6° (1.0% in chloroform) (c) a proton magnetic resonancespectrum in CDCl₃ having principal signals at (s=singlet, d=doublet,t=triplet, m=multiple) 0.83s, 1.15d (J=6), 1.72s, 2.02m, 2.81d (J=4),3.13d (J=4), 3.5-4.0 (complex signals; area=3), 4.80d (J=2), 5.49 broadd (J=6), 5.22-6.02 (complex signals; area=3), 6.08dd (J=4,6) 6.06t(J=11); and 7.60dd (J=11,15) parts per million downfield fromtetramethylsilane (d) a carbon 13 nuclear magnetic resonance spectrum inCDCl₃ having principal signals in parts per million, at:

    ______________________________________                                        Ppm (downfield  Ppm (downfield                                                from TMS)       from TMS)                                                     ______________________________________                                        173.95                  73.46                                                 167.43                  69.74                                                 166.46                  66.83                                                 143.37                  66.34                                                 140.57                  65.59                                                 138.89                  62.51                                                 130.53                  48.86                                                 118.88                  48.00                                                 118.72                  44.28                                                 116.67                  36.19                                                 85.38                   29.23                                                 78.91                   28.04                                                 78.42                   23.19                                                 77.02           CDCl.sub.3                                                                            21.14                                                 75.62                   18.44                                                 75.40                   6.57                                                  ______________________________________                                    


2. A pharmaceutical composition useful for treating bacterial infectionscomprising an effective antibacterial amount of roridin L- 2 accordingto claim 1 and a pharmaceutically acceptable carrier.
 3. A method oftreating bacterial infections in humans by administering an effectiveantibacterial amount of roridin L-2 according to claim 1.